1. Field of the Invention
The present invention relates to stem cells. More specifically, the present invention relates to self-renewing pluripotent hepatic stem cells.
2. Description of the Related Art
The enormous regenerative capacity of the liver after partial hepatectomy or chemical injury is well known. In rodents, liver weight returns to normal within a few weeks even after loss of up to two-thirds of total liver mass (Fausto, N., and E. M. Webber. 1994. Liver regeneration: The liver. I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. A. Schachter, and D. A. Shafritz, editors: Raven Press, N.Y., ed. 3, 1059–1084.). Remarkable regenerative potential is also retained in hematopoiesis. Hematopoietic stem cells(HSCs) certainly exist in bone marrow where they self-renew and differentiate along all hematopoietic lineages. Sophisticated isolation methods have recently identified a highly probable HSC candidate; a single such cell can reconstitute bone marrow (Osawa, M., K. Hanada, H. Hamada, and H. Nakauchi. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273: 242–245). By analogy with hematopoiesis, liver regeneration can be regarded as mediated by proliferation and differentiation of hepatic stem cells. However, it remains unclear how the liver is regenerated and what cells are involved in such regeneration. Overturf et al. (1997) inferred from serial transplantation studies the presence in adult mouse liver of cells capable of dividing more than 60 times; they ascribed this great reconstitutive ability to hepatic stem/progenitor cells (Overturf, K., M Al-Dhalimy, C. N. Ou, M. Finegold, and M. Grompe. 1997. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am. J. Pathol. 151: 1273–1280). However, examination in greater detail strongly indicated that fully differentiated hepatocytes but not progenitors divided intensively after cell transplantation. In addition, the reconstitutive capacity of serially transplanted hepatocytes was as high as that of freshly isolated hepatocytes (Overturf, K., M. Al-Dhalimy, M. Finegold, and M. Grompe. 1999. The repopulation potential of hepatocyte populations differing in size and prior mitotic expansion. Am. J. Pathol. 155: 2135–2143). Of major importance in understanding proliferative processes in the liver is to recognize that fully differentiated hepatocytes themselves possess great growth potential and that stem cells may not be required for liver regeneration (Michalopoulos, G. K., and M. C. DeFrances. 997. Liver regeneration. Science 276: 60–66).
By contrast, it is believed that in the developing liver both hepatocytes and cholangiocytes differentiate from a common cell component, the hepatoblast (Shiojiri, N. 1984. The origin of intrahepatic bile duct cells in the mouse. J. Embryol. Exp. Morphol. 79: 25–39; Shiojiri, N., J. M. Lemire, and N. Fausto. 1991. Cell lineages and oval cell progenitors in rat liver development Cancer Res. 51: 2611–2620; and Fausto, N. 1994. Liver stem cells: The liver. I. M. Arias, J. L. Boyer, N. Fausto, W. B. Jakoby, D. A. Schachter, and D. A. Shafritz, editors: Raven Press, N.Y., ed. 3. 1501–1518). It was reported that fetal rat liver cells transplanted into retrorsine-treated liver reconstituted bile duct and hepatocyte structures (Dabeva, M. D., P. M. Petkov, J. Sandhu, R. Oren, E. Laconi, E. Hurston, and D. A. Shafritz. 2000. Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am. J. Pathol. 156: 2017–2031).This result suggested that the donor cell population included at least bipotent hepatic stem/progenitor cells. However, it was not possible to determine if the regenerated structures had differentiated from stem cells or from lineage-committed cells. Recently, by combining FACS® and in vitro clonal analysis bipotent hepatic progenitor cells have been isolated from rats or mice (Kubota, H., and L. M. Reid. 2000. Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc. Natl. Acad. Sci. USA 97: 12132–12137; Suzuki, A., Y. Zheng, R. Kondo, M. Kusakabe, Y. Takada, K. Fukao, H. Nakauchi, and H. Taniguchi. 2000. Flow cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 32: 1230–1239). Although these data demonstrated that isolated cell is a possible candidate for the hepatic stem cell in the developing liver, its self renewal potential and multiple differentiation capability remain largely unanswered. For this reason, isolated progenitor cells have never been identified as hepatic stem cells.
Stem cells are generally defined as clonogenic cells capable of both self-renewal and multilineage differentiation (Till, J. E., and E. A. McCulloch. 1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14: 1419–1430; Metcalf, D., and M. A. S. Moore. 1971. Hematopoietic Cells (Amsterdam. North-Holland)). During development and regeneration of a given tissue, such cells give rise to non self-renewing progenitors with restricted differentiation potential and finally to functionally mature cells. In the study reported here, using in vitro single cell-based assays, we further enriched candidate hepatic stem cells and clonogenically identified cells with self-renewing capability and multilineage differentiation potential. These cells could be clonally propagated in culture for >6 months where they continuously produced hepatocytes and cholangiocytes as descendants. The value of stem cells expanded in vitro is expected to be great not only in conventional studies of their differentiation or self-renewing potential but also in therapy, for example, with virus-mediated gene transfer or as theoretically unlimited sources of cells Furthermore, upon cell transplantation these cells differentiated not only into hepato-biliary lineage cells but cells in other organs of endodermorigin such as pancreas and intestine. Thus, the stem cells that we isolated may actually be primitive endodermalstem cells persisting in fetal mouse liver. Alternatively, these findings may reflect lineage plasticity or transdifferentiation of hepatic stem cells.
We have previously reported that cells in ED 13.5 fetal mouse liver cells which co-express CD49f and CD29 (α6 and β1 integrin subunits) but do not express c-Kit (stem cell factor receptor), CD45 (leukocyte common antigen), or TER119 (a molecule resembling glycophorin and exclusively expressed on immature erythroid cells) are the best candidate hepatic stem/progenitor cells (Suzuki et al., 2000).
Previously, there have been no cloned hepatic stem cells reported which are capable of renewing and multiply differentiating into a variety of cells. Therefore, there remains need for providing such cloned hepatic stem cells which are capable of self-renewing and possesse pluripotency, e, g., for use in the regenerative therapy, particularly for treating liver diseases and disorders.
The object of the present invention is thus to provide such self-renewing and pluripotent cells.